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Effect of extracellular divalent cations on P2X7 receptor-mediated IL-1β release FROM THP-1 human monocytic cells The P2X7 receptor is an ATP-gated ion channel widely expressed in immune cells. Activation of P2X7 receptors by either ATP or Benzoyl-Benzoyl ATP (BzATP) in lipopolysaccharide (LPS) treated monocytes or macrophages consistently causes release of mature interleukin-1beta (mIL-1β) although there are conflicting reports on the role of extracellular calcium in this response ( Gudipathy et al., 2003, Brough et al.,2003, Andrei et al., 2004). A potential complicating factor in these studies is the measurement of IL-1β using antibodies that may not clearly differentiate between the mature and immature forms of IL-1β . We have previously (Buell et al., 1998) described a reporter gene assay that only detects mature IL-1β (mIL-1β) and so the aim of this study was to re-examine the role of extracellular calcium on P2X 7-mediated mIL-1β release in THP-1 monocytes, which express P2X7 receptors endogenously. Agonist stimulated mIL-1β release from THP-1 monocytes was measured as described (Buell et al., 1998) in the presence of varying concentrations of calcium or magnesium in the assay buffer (140mM NaCl/10mM Hepes/5mM KCl/10mM glucose, pH7.4). Cell viability was measured using a commercially available LDH release assay. All data are the mean and S.E.M of 3 experiments. ATP and BzATP released mIL-1β in both the absence and presence of calcium (no magnesium in assay buffer). Maximal responses to BzATP and ATP were obtained at 0.1mM calcium, but in the absence of calcium appreciable mIL-1β release occurred (BzATP 66 ± 4% and ATP 74 ± 24% of max response in presence of 0.1mM calcium). The effect of varying extracellular calcium was to change agonist potency (pEC50 for BzATP were 4.9 ± 0.11, 4.5 ± 0.09 and 3.7 ± 0.05 at 0, 0.1 and 1mM calcium respectively). Similar results were obtained when magnesium concentration was varied in the extracellular media in the complete absence of calcium. BzATP also induced cell death in these studies (i.e. 27 ± 11% total LDH at 256 μM BzATP). The agonist potency for eliciting this response was similar to that for stimulating mIL-1β release. In summary, neither extracellular Ca2+ nor Mg 2+ were required for P2X 7-receptor mediated release of mIL-1β in our THP-1 cells. Indeed, divalent cations reduced agonist potency which is consistent with their actions in other studies on the P2X7 receptor (Michel et al., 1999). The reasons for the difference in calcium sensitivity of IL-1β release between studies is not known but this study suggests that extracellular calcium is not universally required to support P2X7 receptor mediated IL-1β release.
Andrei et al., (2004), PNAS, 101, 9745-9750. |
